Small molecule therapeutics for neuroinflammation-mediated neurodegenerative disorders.

Chronically activated microglia and the resulting cascade of neuroinflammatory mechanisms have been postulated to play a critical role in neurodegenerative disorders. Microglia are the main component of the brain's innate immune system and become activated by infection, injury, misfolded proteins or a multitude of other stimuli. Activated microglia release pro-inflammatory and cytotoxic factors that can damage neurons and transform astrocytes to become toxic to neurons as well. Therapeutic approaches aiming to modulate microglia activation may be beneficial to mitigate the progression of inflammatory-mediated neurodegenerative diseases. In this literature review, we provide an overview of recent progress on key microglia targets and discovery of small molecule compounds advancing in clinical trials to minimize neuroinflammation.

Effects of CYP46A1 Inhibition on Long-Term-Depression in Hippocampal Slices ex vivo and 24S-Hydroxycholesterol Levels in Mice in vivo.

The manipulation of cholesterol and its metabolites has been hypothesized to be therapeutically beneficial for mood disorders, neurodegenerative disorders, and epilepsies. A major regulator of cholesterol clearance and turnover in the central nervous system is CYP46A1, a brain enriched enzyme responsible for metabolism of cholesterol into 24S-hydroxycholesterol. Inhibition of this enzyme may negatively modulate NMDARs as 24S-hydroxycholesterol was shown to enhance NMDAR function. In addition, alterations of local cholesterol or other changes mediated by CYP46A1 activity could have important influences on central nervous system function. Here we demonstrate that humans and mice display brain region specific and similar CYP46A1 and 24S-hydroxycholesterol distribution. Treatment with distinct classes of CYP46A1 inhibitors led to central 24S-hydroxycholesterol reduction in vivo and ablation of long term depression in hippocampal slices. Our results suggest that rodents show similarity to humans for studying the impact of CYP46A1 inhibitors and that rapid, local modulation of oxysterols can be achieved through CYP46A1 inhibition.

PAC1 receptor blockade reduces central nociceptive activity: new approach for primary headache?

Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) may play an important role in primary headaches. Preclinical evidence suggests that PACAP38 modulates trigeminal nociceptive activity mainly through PAC1 receptors while clinical studies report that plasma concentrations of PACAP38 are elevated in spontaneous attacks of cluster headache and migraine and normalize after treatment with sumatriptan. Intravenous infusion of PACAP38 induces migraine-like attacks in migraineurs and cluster-like attacks in cluster headache patients. A rodent-specific PAC1 receptor antibody Ab181 was developed, and its effect on nociceptive neuronal activity in the trigeminocervical complex was investigated in vivo in an electrophysiological model relevant to primary headaches. Ab181 is potent and selective at the rat PAC1 receptor and provides near-maximum target coverage at 10 mg/kg for more than 48 hours. Without affecting spontaneous neuronal activity, Ab181 effectively inhibits stimulus-evoked activity in the trigeminocervical complex. Immunohistochemical analysis revealed its binding in the trigeminal ganglion and sphenopalatine ganglion but not within the central nervous system suggesting a peripheral site of action. The pharmacological approach using a specific PAC1 receptor antibody could provide a novel mechanism with a potential clinical efficacy in the treatment of primary headaches.

Effects of monoclonal antagonist antibodies on calcitonin gene-related peptide receptor function and trafficking.

BACKGROUND: Monoclonal antibodies against calcitonin gene-related peptide (CGRP) or its receptor are efficacious for the prevention of migraine headaches. The downstream molecular mechanisms following ligand-receptor blockade by which these antibodies prevent CGRP signaling through CGRP receptors have not been demonstrated. METHODS: Here we produced tool monoclonal functional antagonist antibodies against CGRP and its canonical receptor and developed a novel cellular model using fluorogen-activated protein technology that allows detection of CGRP receptor internalization by flow cytometry and, for an extended time course, visualization by confocal microscopy. RESULTS: Using this cell model we showed that these antagonist antibodies block both CGRP-induced cAMP signaling and CGRP receptor internalization. At least 10-fold higher concentrations of either antibody are necessary to block CGRP receptor internalization compared with cAMP accumulation in our cell model. CONCLUSION: These data reinforce our understanding of how monoclonal functional antagonist antibodies interfere with CGRP signaling.

Therapeutic approaches to enhance PINK1/Parkin mediated mitophagy for the treatment of Parkinson's disease.

The discovery of rare familial monogenic forms of early-onset Parkinson's disease has led to the identification of a mitochondrial quality control process as a key player in this disease. Loss-of-function mutations in the genes encoding PINK1 or Parkin result in insufficient removal of dysfunctional mitochondria through autophagy, a process termed mitophagy. Understanding the mechanism of this process and the function of its two key players, PINK1 and Parkin, has led to the discovery of new therapeutic approaches. Small molecule activators of mitophagy, either activating PINK1 or Parkin directly or inhibiting Parkin's counterplayer, the ubiquitin-specific protease USP30, are in preclinical development. To enable clinical success of future small molecule mitophagy enhancers, biomarkers for mitochondrial integrity and mitophagy are being developed. Only a few years after the discovery of mitophagy deficits in Parkinson's disease, research of the underlying mechanisms, drug discovery of modulators for this mechanism and identification of biomarkers provide new avenues towards the development of disease-modifying therapies.

PAC1 receptor mRNA and protein distribution in rat and human trigeminal and sphenopalatine ganglia, spinal trigeminal nucleus and in dura mater.

BACKGROUND: To further understand the role of pituitary adenylate cyclase-activating polypeptide 1 (PAC1) receptors in headache disorders, we mapped their expression in tissues of the trigemino-autonomic system by immunohistochemistry and in situ hybridization. METHODS: To optimize screening for monoclonal antibodies suitable for immunohistochemistry on formalin-fixed, paraffin-embedded tissues, we developed a new enzyme-linked immunosorbent assay using formalin-fixed, paraffin-embedded cells overexpressing human PAC1 receptors. 169G4.1 was selected from these studies for analysis of rat and human tissues and chimerized onto a mouse backbone to avoid human-on-human cross-reactivity. Immunoreactivity was compared to PAC1 receptor mRNA by in situ hybridization in both species. RESULTS: 169G4.1 immunoreactivity delineated neuronal cell bodies in the sphenopalatine ganglion in both rat and human, whereas no staining was detected in the trigeminal ganglion. The spinal trigeminal nucleus in both species showed immunoreactivity as especially strong in the upper laminae with both cell bodies and neuropil being labelled. No immunoreactivity was seen in either rat or human dura mater vessels. In situ hybridization in both species revealed mRNA in sphenopalatine ganglion neurons and the spinal trigeminal nucleus, a weak signal in the trigeminal nucleus and no signal in dural vessels. CONCLUSION: Taken together, these data support a role for PAC1 receptors in the trigemino-autonomic system as it relates to headache pathophysiology.

Long-acting MIC-1/GDF15 molecules to treat obesity: Evidence from mice to monkeys.

In search of metabolically regulated secreted proteins, we conducted a microarray study comparing gene expression in major metabolic tissues of fed and fasted ob/ob mice and C57BL/6 mice. The array used in this study included probes for ~4000 genes annotated as potential secreted proteins. Circulating macrophage inhibitory cytokine 1 (MIC-1)/growth differentiation factor 15 (GDF15) concentrations were increased in obese mice, rats, and humans in comparison to age-matched lean controls. Adeno-associated virus-mediated overexpression of GDF15 and recombinant GDF15 treatments reduced food intake and body weight and improved metabolic profiles in various metabolic disease models in mice, rats, and obese cynomolgus monkeys. Analysis of the GDF15 crystal structure suggested that the protein is not suitable for conventional Fc fusion at the carboxyl terminus of the protein. Thus, we used a structure-guided approach to design and successfully generate several Fc fusion molecules with extended half-life and potent efficacy. Furthermore, we discovered that GDF15 delayed gastric emptying, changed food preference, and activated area postrema neurons, confirming a role for GDF15 in the gut-brain axis responsible for the regulation of body energy intake. Our work provides evidence that GDF15 Fc fusion proteins could be potential therapeutic agents for the treatment of obesity and related comorbidities.

Does inactivation of USP14 enhance degradation of proteasomal substrates that are associated with neurodegenerative diseases?

A common pathological hallmark of age-related neurodegenerative diseases is the intracellular accumulation of protein aggregates such as α-synuclein in Parkinson's disease, TDP-43 in ALS, and tau in Alzheimer's disease. Enhancing intracellular clearance of aggregation-prone proteins is a plausible strategy for slowing progression of neurodegenerative diseases and there is great interest in identifying molecular targets that control protein turnover. One of the main routes for protein degradation is through the proteasome, a multisubunit protease that degrades proteins that have been tagged with a polyubiquitin chain by ubiquitin activating and conjugating enzymes. Published data from cellular models indicate that Ubiquitin-specific protease 14 (USP14), a deubiquitinating enzyme (DUB), slows the degradation of tau and TDP-43 by the proteasome and that an inhibitor of USP14 increases the degradation of these substrates. We conducted similar experiments designed to evaluate tau, TDP-43, or α-synuclein levels in cells after overexpressing USP14 or knocking down endogenous expression by siRNA.

Immunohistochemical localization of the calcitonin gene-related peptide binding site in the primate trigeminovascular system using functional antagonist antibodies.

Calcitonin gene-related peptide (CGRP) is a potent vasodilator and a neuromodulator implicated in the pathophysiology of migraine. It binds to the extracellular domains of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) 1 that together form the CGRP receptor. Antagonist antibodies against CGRP and its binding site at the receptor are clinically effective in preventing migraine attacks. The blood-brain barrier penetration of these antagonist antibodies is limited, suggesting that a potential peripheral site of action is sufficient to prevent migraine attacks. To further understand the sites of CGRP-mediated signaling in migraine, we used immunohistochemical staining with recently developed antagonist antibodies specifically recognizing a fusion protein of the extracellular domains of RAMP1 and CLR that comprise the CGRP binding pocket at the CGRP receptor in monkey and man. We confirmed binding of the antagonist antibodies to human vascular smooth muscle cells (VSMCs) of dural meningeal arteries and neurons in the trigeminal ganglion, both of which are likely sites of action for therapeutic antibodies in migraine patients. We further used one of these antibodies for detailed mapping on cynomolgus monkey tissue and found antagonist antibody binding sites at multiple levels in the trigeminovascular system: in the dura mater VSMCs, in neurons and satellite glial cells in the trigeminal ganglion, and in neurons in the spinal trigeminal nucleus caudalis. These data reinforce and clarify our understanding of CGRP receptor localization in a pattern consistent with a role for CGRP receptors in trigeminal sensitization and migraine pathology.

Radiosynthesis and initial characterization of a PDE10A specific PET tracer [18F]AMG 580 in non-human primates.

INTRODUCTION: Phosphodiesterase 10A (PDE10A) is an intracellular enzyme responsible for the breakdown of cyclic nucleotides which are important second messengers for neurotransmission. Inhibition of PDE10A has been identified as a potential target for treatment of various neuropsychiatric disorders. To assist drug development, we have identified a selective PDE10A positron emission tomography (PET) tracer, AMG 580. We describe here the radiosynthesis of [(18)F]AMG 580 and in vitro and in vivo characterization results. METHODS: The potency and selectivity were determined by in vitro assay using [(3)H]AMG 580 and baboon brain tissues. [(18)F]AMG 580 was prepared by a 1-step [(18)F]fluorination procedure. Dynamic brain PET scans were performed in non-human primates. Regions-of-interest were defined on individuals' MRIs and transferred to the co-registered PET images. Data were analyzed using two tissue compartment analysis (2TC), Logan graphical (Logan) analysis with metabolite-corrected input function and the simplified reference tissue model (SRTM) method. A PDE10A inhibitor and unlabeled AMG 580 were used to demonstrate the PDE10A specificity. KD was estimated by Scatchard analysis of high and low affinity PET scans. RESULTS: AMG 580 has an in vitro KD of 71.9 pM. Autoradiography showed specific uptake in striatum. Mean activity of 121 ± 18 MBq was used in PET studies. In Rhesus, the baseline BPND for putamen and caudate was 3.38 and 2.34, respectively, via 2TC, and 3.16, 2.34 via Logan, and 2.92, and 2.01 via SRTM. A dose dependent decrease of BPND was observed by the pre-treatment with a PDE10A inhibitor. In baboons, 0.24 mg/kg dose of AMG 580 resulted in about 70% decrease of BPND. The in vivo KD of [(18)F]AMG 580 was estimated to be around 0.44 nM in baboons. CONCLUSION: [(18)F]AMG 580 is a selective and potent PDE10A PET tracer with excellent specific striatal binding in non-human primates. It warrants further evaluation in humans.

AMG 580: a novel small molecule phosphodiesterase 10A (PDE10A) positron emission tomography tracer.

Phosphodiesterase 10A (PDE10A) inhibitors have therapeutic potential for the treatment of psychiatric and neurologic disorders, such as schizophrenia and Huntington's disease. One of the key requirements for successful central nervous system drug development is to demonstrate target coverage of therapeutic candidates in brain for lead optimization in the drug discovery phase and for assisting dose selection in clinical development. Therefore, we identified AMG 580 [1-(4-(3-(4-(1H-benzo[d]imidazole-2-carbonyl)phenoxy)pyrazin-2-yl)piperidin-1-yl)-2-fluoropropan-1-one], a novel, selective small-molecule antagonist with subnanomolar affinity for rat, primate, and human PDE10A. We showed that AMG 580 is suitable as a tracer for lead optimization to determine target coverage by novel PDE10A inhibitors using triple-stage quadrupole liquid chromatography-tandem mass spectrometry technology. [(3)H]AMG 580 bound with high affinity in a specific and saturable manner to both striatal homogenates and brain slices from rats, baboons, and human in vitro. Moreover, [(18)F]AMG 580 demonstrated prominent uptake by positron emission tomography in rats, suggesting that radiolabeled AMG 580 may be suitable for further development as a noninvasive radiotracer for target coverage measurements in clinical studies. These results indicate that AMG 580 is a potential imaging biomarker for mapping PDE10A distribution and ensuring target coverage by therapeutic PDE10A inhibitors in clinical studies.

Synthesis and preliminary biological evaluation of potent and selective 2-(3-alkoxy-1-azetidinyl) quinolines as novel PDE10A inhibitors with improved solubility.

We report the discovery of a novel series of 2-(3-alkoxy-1-azetidinyl) quinolines as potent and selective PDE10A inhibitors. Structure-activity studies improved the solubility (pH 7.4) and maintained high PDE10A activity compared to initial lead compound 3, with select compounds demonstrating good oral bioavailability. X-ray crystallographic studies revealed two distinct binding modes to the catalytic site of the PDE10A enzyme. An ex vivo receptor occupancy assay in rats demonstrated that this series of compounds covered the target within the striatum.

Antibodies to the extracellular pore loop of TRPM8 act as antagonists of channel activation.

The mammalian transient receptor potential melastatin channel 8 (TRPM8) is highly expressed in trigeminal and dorsal root ganglia. TRPM8 is activated by cold temperature or compounds that cause a cooling sensation, such as menthol or icilin. TRPM8 may play a role in cold hypersensitivity and hyperalgesia in various pain syndromes. Therefore, TRPM8 antagonists are pursued as therapeutics. In this study we explored the feasibility of blocking TRPM8 activation with antibodies. We report the functional characterization of a rabbit polyclonal antibody, ACC-049, directed against the third extracellular loop near the pore region of the human TRPM8 channel. ACC-049 acted as a full antagonist at recombinantly expressed human and rodent TRPM8 channels in cell based agonist-induced 45Ca2+ uptake assays. Further, several poly-and monoclonal antibodies that recognize the same region also blocked icilin activation of not only recombinantly expressed TRPM8, but also endogenous TRPM8 expressed in rat dorsal root ganglion neurons revealing the feasibility of generating monoclonal antibody antagonists. We conclude that antagonist antibodies are valuable tools to investigate TRPM8 function and may ultimately pave the way for development of therapeutic antibodies.

Discovery of Novel Imidazo[4,5-b]pyridines as Potent and Selective Inhibitors of Phosphodiesterase 10A (PDE10A).

We report the discovery of novel imidazo[4,5-b]pyridines as potent and selective inhibitors of PDE10A. The investigation began with our recently disclosed ketobenzimidazole 1, which exhibited single digit nanomolar PDE10A activity but poor oral bioavailability. To improve oral bioavailability, we turned to novel scaffold imidazo[4,5-b]pyridine 2, which not only retained nanomolar PDE10A activity but was also devoid of the morpholine metabolic liability. Structure-activity relationship studies were conducted systematically to examine how various regions of the molecule impacted potency. X-ray cocrystal structures of compounds 7 and 24 in human PDE10A helped to elucidate the key bonding interactions. Five of the most potent and structurally diverse imidazo[4,5-b]pyridines (4, 7, 12b, 24a, and 24b) with PDE10A IC50 values ranging from 0.8 to 6.7 nM were advanced into receptor occupancy studies. Four of them (4, 12b, 24a, and 24b) achieved 55-74% RO at 10 mg/kg po.

Comparison of phosphodiesterase 10A, dopamine receptors D1 and D2 and dopamine transporter ligand binding in the striatum of the R6/2 and BACHD mouse models of Huntington's disease.

BACKGROUND: Phosphodiesterase 10A (PDE10A) is expressed at high levels in the striatum and has been proposed both as a biomarker for Huntington's disease pathology and as a target for intervention. OBJECTIVE: PDE10A radiotracers have been successfully used to measure changes in binding density in Huntington's disease patients, but little is known about PDE10A binding in mouse models that are used extensively to model pathology and test therapeutic interventions. METHODS: Our study investigated changes in PDE10A binding using the selective tracer 3H-7980 at specific ages of two Huntington's disease transgenic mouse models: R6/2, a short-lived model carrying exon-1 of mutant HTT and BACHD, a longer-lived model carrying full-length mutant HTT. PDE10A binding was compared to binding of known markers of striatal atrophy in Huntington's disease, e.g. dopamine transporter (DAT) and dopamine receptors D1 and D2. RESULTS: We found that in the R6/2 model at 6 weeks of age, mice showed high variability of binding, however binding of all ligands was significantly decreased at 8 and 12 weeks of age. In contrast, no changes were detectable in the BACHD model at 8, 10 or 12 month of age. CONCLUSIONS: These findings suggest that radiotracer binding of PDE10A, DAT, D1 and D2 receptor in the R6/2 model may be a good indicator of striatal pathological changes that are observed in Huntington's disease patients, and that the first 12 months in the BACHD model may be more reflective of early stages of the disease.

Antidepressant and anxiolytic potential of the multimodal antidepressant vortioxetine (Lu AA21004) assessed by behavioural and neurogenesis outcomes in mice.

Vortioxetine (Lu AA21004) is an investigational novel antidepressant with multimodal activity that functions as a 5-HT3, 5-HT7 and 5-HT(1D) receptor antagonist, 5-HT(1B) receptor partial agonist, 5-HT(1A) receptor agonist and inhibitor of the 5-HT transporter in vitro. Here we explore its anxiolytic and antidepressant potential in adult mice. Vortioxetine was assessed in BalB/cJ@RJ mice using the open-field and forced-swim tests (acute: p.o. 1 h, repeated: daily p.o. 21 days), and in 129S6/SvEvTac mice using the novelty suppressed feeding paradigm (acute: p.o. 1 h, sustained: daily p.o. 14 or 21 days). Fluoxetine and diazepam were controls. Acute and repeated dosing of vortioxetine produced more pronounced anxiolytic- and antidepressant-like activities than fluoxetine. Vortioxetine significantly increased cell proliferation and cell survival and stimulated maturation of immature granule cells in the subgranular zone of the dentate gyrus of the hippocampus after 21 days of treatment. After 14 days, a high dose of vortioxetine increased dendritic length and the number of dendrite intersections, suggesting that vortioxetine accelerates the maturation of immature neurons. Vortioxetine displays an antidepressant and anxiolytic profile following repeated administration associated with increased neurogenesis at several stages. Vortioxetine effects were observed at low levels of 5-HT transporter occupancy, suggesting an alternative mechanism of action to 5-HT reuptake inhibition.

Regional, kinetic [(18)F]FDG PET imaging of a unilateral Parkinsonian animal model.

Positron emission tomography (PET) imaging with the glucose analog 2-deoxy-2-[(18)F]fluoro-D-glucose ([(18)F] FDG) has demonstrated clinical utility for the monitoring of brain glucose metabolism alteration in progressive neurodegenerative diseases. We examined dynamic [(18)F]FDG PET imaging and kinetic modeling of atlas-based regions to evaluate regional changes in the cerebral metabolic rate of glucose in the widely-used 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. Following a bolus injection of 18.5 ± 1 MBq [(18)F]FDG and a 60-minute PET scan, image-derived input functions from the vena cava and left ventricle were used with three models, including Patlak graphical analysis, to estimate the influx constant and the metabolic rate in ten brain regions. We observed statistically significant changes in [(18)F]FDG uptake ipsilateral to the 6-OHDA injection in the basal ganglia, olfactory bulb, and amygdala regions; and these changes are of biological relevance to the disease. These experiments provide further validation for the use of [(18)F]FDG PET imaging in this model for drug discovery and development.

Vortioxetine (Lu AA21004), a novel multimodal antidepressant, enhances memory in rats.

The serotonergic system plays an important role in cognitive functions via various 5-HT receptors. Vortioxetine (Lu AA21004) in development as a novel multimodal antidepressant is a 5-HT3, 5-HT7 and 5-HT1D receptor antagonist, a 5-HT1B receptor partial agonist, a 5-HT1A receptor agonist and a 5-HT transporter (5-HTT) inhibitor in vitro. Preclinical studies suggest that 5-HT3 and 5-HT7 receptor antagonism as well as 5-HT1A receptor agonism may have a positive impact on cognitive functions including memory. Thus vortioxetine may potentially enhance memory. We investigated preclinical effects of vortioxetine (1-10mg/kg administered subcutaneously [s.c.]) on memory in behavioral tests, and on cortical neurotransmitter levels considered important in rat memory function. Contextual fear conditioning and novel object recognition tests were applied to assess memory in rats. Microdialysis studies were conducted to measure extracellular neurotransmitter levels in the rat medial prefrontal cortex. Vortioxetine administered 1h before or immediately after acquisition of contextual fear conditioning led to an increase in freezing time during the retention test. This mnemonic effect was not related to changes in pain sensitivity as measured in the hotplate test. Rats treated with vortioxetine 1h before training spent more time exploring the novel object in the novel object recognition test. In microdialysis studies of the rat medial prefrontal cortex, vortioxetine increased extracellular levels of acetylcholine and histamine. In conclusion, vortioxetine enhanced contextual and episodic memory in rat behavioral models. Further demonstration of its potential effect on memory functions in clinical settings is warranted.

Rapid identification of a novel small molecule phosphodiesterase 10A (PDE10A) tracer.

A radiolabeled tracer for imaging therapeutic targets in the brain is a valuable tool for lead optimization in CNS drug discovery and for dose selection in clinical development. We report the rapid identification of a novel phosphodiesterase 10A (PDE10A) tracer candidate using a LC-MS/MS technology. This structurally distinct PDE10A tracer, AMG-7980 (5), has been shown to have good uptake in the striatum (1.2% ID/g tissue), high specificity (striatum/thalamus ratio of 10), and saturable binding in vivo. The PDE10A affinity (K(D)) and PDE10A target density (B(max)) were determined to be 0.94 nM and 2.3 pmol/mg protein, respectively, using [(3)H]5 on rat striatum homogenate. Autoradiography on rat brain sections indicated that the tracer signal was consistent with known PDE10A expression pattern. The specific binding of [(3)H]5 to rat brain was blocked by another structurally distinct, published PDE10A inhibitor, MP-10. Lastly, our tracer was used to measure in vivo PDE10A target occupancy of a PDE10A inhibitor in rats using LC-MS/MS technology.

Characterization of [³H]Lu AE60157 ([³H]8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) binding to 5-hydroxytryptamine6 (5-HT6) receptors in vivo.

The serotonin6 (5-HT(6)) receptor has received attention for its proposed role in cognitive impairments associated with schizophrenia and Alzheimer's disease. This has lead to a search for selective 5-HT(6) receptor ligands useful for in vivo imaging in animals and humans. The novel 5-HT(6) receptor antagonist Lu AE60157 (8-(4-methylpiperazin-1-yl)-3-phenylsulfonylquinoline) displays high affinity for the human (h) 5-HT(6) receptor (K(d) 0.2nM), and broad profiling in 60 additional binding and enzyme assays showed that Lu AE60157 displays 16-fold selectivity to the h5-HT(2A) receptor (K(i) 3.2nM) and >100-fold selectivity to all other evaluated targets. Lu AE60157 was labeled with tritium in the N-methyl group and evaluated as a radioligand in vitro as well as in vivo in rats and mice. Autoradiography experiments showed that [(3)H]Lu AE60157 bound preferentially to rat brain regions with expected high 5-HT(6) receptor density. Furthermore, [(3)H]Lu AE60157 showed good brain penetration after systemic administration and high (about 75%) specific in vivo binding to the striatal 5-HT(6) receptor in rats. The striatal binding of [(3)H]Lu AE60157 was fully displaced by selective 5-HT(6) receptor antagonists (SB-742457; Lu AE58054) and antipsychotics known to inhibit the binding of 5-HT(6) receptors in vitro (clozapine; olanzapine; sertindole), but was not displaced by antipsychotics lacking high 5-HT(6) receptor affinities (risperidone; haloperidol; quetiapine). No specific binding to mouse brain tissue in vivo could be obtained. In conclusion, [(3)H]Lu AE60157 is suitable for measuring in vivo occupancies of 5-HT(6) receptor ligands in rat brain regions in which 5-HT(2A) receptors do not interfere.